Selective deuteration of tyrosine, aspartic and glutamic acids

ABSTRACT

A method for the preparation of deuterated L-tyrosine, Laspartic acid and L-glutamic acid which comprises treating protonated L-tyrosine, L-aspartic acid or L-glutamic acid or a suitable partially deuterated analog thereof with either a deuterated inorganic acid or an alkali metal deuteroxide or alkaline earth metal deuteroxide. The products thus obtained are useful as intermediates in the preparation and study of labelled peptides, polypeptides and proteins and as a growth medium for biological investigations.

United States Patent Putter 541 SELECTIVE'DEUTERATION 0F TYROSINE,ASPARTIC AND GLUTAMIC ACIDS [72] Inventor: Irving Putter, Martinsville,NJ.

[73] Assignee: Merck & Co., Inc., Rahway, NJ.

[22] Filed: Aug. 25, 1969 [2]] Appl. No.: 852,926

[52] US. Cl. ..260/519, 260/534 E, 260/534 G [51] Int. Cl ..C07c 101/04[58] Field of Search ..260/5 I9, 534 E, 534 G [56] References CitedOTHER PUBLICATIONS Remy, I-I., Treatise on Inorganic Chemistry, Vol.-II, (1956), pub. by Elsevier Pub, Co., New York, pp. 558 and 559 reliedon.

[451 Oct. 17,1972

Primary Examiner-Lorraine A. Weinberger Assistant Examiner-14. ArnoldThaxton Attorney-Joseph W. Molasky, J. Jerome Behan and I. Louis Wolk [57 ABSTRACT A method for the preparation of deuterated L- tyrosine,L-aspartic acid and L-glutamic acid which comprises treating protonatedL-tyrosine, L-aspartic acid or L-glutamic acid or a suitable partiallydeuterated analog thereof with either a deuterated inorganic acid or analkali metal deuteroxide or alkaline earth metal deuteroxide. Theproducts thus obtained are useful as intermediates in the preparationand study of labelled peptides, polypeptides and proteins and as agrowth medium for biological investigations.

25 Claims, No Drawings l and SELECTIVE DEUTERATION OF TYROSINE, ASPARTICAND GLUTAMIC ACIDS Thisinvention relates to a novel class of deuteratedproducts selected from deuterated L-tyrosine, L-aspartic acidandL-glutamic acid and ma novel method for theirpreparation.Specifically, the novel deuterated L- tyrosine,'L-aspartic acid andL-glutarnic acid products I of this invention are compounds having thefollowing wherein R-is hydrogen or deuterium and n is aninteger having avalue ofO ml. 1

' The-products ofthis invention are usefulin preparing "syntheticallylabelled peptides and 'in fermentation processes-in affording labelledproteins. In addition, the instant products have utility as a growthmedium-in the preparation of Staphylococcus Aureus bacteria from whichthere can beisolated a deuterated staphylococcal nuclease, w hich enzymeis useful in thestudy of the mechanism of the action of an enzyme on itssubstrate by nuclear magnetic resonance spectroscopy.

According to the process of this invention the nuclear carbons ortho' tothe hydroxyradical in L- tyrosine, the beta carbon in L-aspartic acidand the gammacarbon in L-glutamic acid are selectively deuterated viathe reaction of .L-tyrosine, L-aspartic acid or L-glutamic acidwith adeuterated inorganic acid as, forexample, with a deuterated mineral acidsuch as deuterium chloride ordeuteriu m bromide or deuterium sulfate,i.e., D580 or, with a deuterated metal hydrox- I ide such as analkalimetal deuteroxide or alkalineearth 'metal deuteroiiid'e as, forexample, with" sodium deuteroxide potassium deuteroxide, calciumdeuteroxide 'or magnesiumdeutetbxide and the like. The concentration ofthe deuterated inorganic acid and deutroxide are not-particularlycritical but,-in practice,'it is most desirable to employ the deuteratedinorganic acid .in-aconcentration offrom about I N to about 7 N, and to.employ the deuterox ide in. a concentrationof from about 0.1 Nto about1.0 N. The application of heat serves to catalyze 'the synthesis and,therefore, in general, it is also desirable to conduct the process atthe reflux temperature of the reactionmixture'or at temperatures in therange of from about 80-l20 C., usually over an extended period of fromabout one hour to several days. The L-tyrosine, L-aspartic acid andL-glutarnic vacid I starting materials of the instant process may be.employed in either their protonated form to afford the partiallydeuterated products described infra asl and la, or, if a completelydeuterated product is desired, the latter may be obtained by simplyemploying the appropriate partially deu-. terate'd precursor (ll andlll, infra). This invention is il lustrated by the following equationsin which A and B depict the formation of partially deuteratedL-tyrosine, L-aspartic acid and L-glutamic acid and equations C and Dillustrate the formation of the corresponding completely deuteratedanalogs (lb and lc, infra).The

2. deuterium. reagent described in the following equations isfdeuteriumchloride, however, this is for illustration only and it should beunderstood that all of the deuterated inorganic acids and alkali metaldeuteroxides and alkaline earth metal deuteroxides mentioned above maybe substituted thereforin an otherwise analogous I process toafford anidentical product:

wherein n is an integer having a value of 0 or l. The followingequations illustrate the preparation of completely deuterated productsaccording to this invention via the deuteration of their correspondingpartially deuterated precursors:

NH: III

D00 o-om-(opi)..-ono 001) .In N03),

wherein n is as defined above. It will be noted in respect of theforegoing equations C and D'that complete deuteration is effected inessentially the same manner as is the deuteration synthesis illustratedby equations A and B because it is the hydrogen atoms bonded to the aconcentratedsolution of an acid followed by lyophilization of-jtheresulting partially deuterated intermediate lll, infraij from deuteriumoxide to effect an exchange of deuterium :for the hydrogen atomsib ondedto the amino and carbonyloxy moieties: Suitable acids 7 :whichmaybetemployed in the process include, for examplepthe mineral acidssuch as hydrochloric; acid,

hydrobromic acid, sulfuric acid andthe like in concentrations of fromabout-l N to 7 N, but, preferably, at a concentrationof about 6 N. Thefollowing equation illustrates this method of preparation:

. wherein n is asdefined above.

, are thenrecovered by elution as, forexample, by'treat deuterated'L-amino .acid mixture thus obtained is then vafford the mentwith asuitably concentrated solution of ammonium hydroxide followed by thefiltration of the concentrate in water I and decolorizat'ion with"charcoal,

' whereafter,;th e resulting mixtureis filtered .a second time and then'concentrated andlyophilized to afford a mixture of the L-amino acidsinideuterate'd form. The

exchange resinand the individual Lramin'o acids are eluted byautomaticallycollecting the effluent while, at

l the same time, a very minimal-quantity as, forexample,

- one-six-hundredth of the effluent; is diverted toan The partiallydeuterated L -tyrosine, L-aspartic acid and L glutamic acidstartingmaterials (ll and Ill)-of the instant: process are obtained viaa m'ulti-step synthesis which comprises subjecting'deuterated algae-to aseries 'of hydrolysis,concentration, filtration,'adsorption andisolation steps. According to this procedure lyophilized deuteratedalgae is treated with a a suitable acid as, for

example, with" a 'trihaloacetic acid such "trifluoroaceticacid ortrichlor'oa'cetic acid ata concentration of fromabo'utv C. to 80 C. orwith dilute vmineral acid as, f or'ex'ample, withaO.l N to 2.0 N solution of hydrochloric acid or sulfuric acid and the like at a temperaturein the range of from about C. to 90 ;C. for a periodof from about 15minutes to 24 hours,

thereby causing the proteins and chlorophyll to separate'fr'om th'ecellsin aninsoluble form-while solubilizing certain undesirable components ofthe algae, ysu chassugars. The insoluble residue obtained bycentrifugation contains-the liberated proteins, chlorophyll" and lipids;By subjecting this residue to a few washes with an; ethanol-ethylether-:mixture whereby the chlorophyllandllipids are solubilized, theresidue containing the liberated protein is obtained. The L-amino acidsthus obtained are then liberated from the protein soluble .polymericmaterial are precipitatedand the acid is removed from the: filtratetoprovide a further concentrate of the amino acids. Elimination of theexcess acid is carried out by concentration to a syrupan d vacuum-dryingin the presence of a suitable base as, for

example, in the presence of an alkali metalhydroxide such as sodiumhydroxide,and dissolving the residue in water andfiltering' off anyresidue'of humin and insoluble polymers.

The. filtrate obtained according to the foregoing'i' procedure is'thenitreatedwith an ion-exchange resin su h' as a sulfonic acid resinwhereby substantially all of 5 v the deuterated L arnino' acids areadsorbed and the amino acid automatic'analyzer .whereby the eluted.amino acid is automatically monitored. The apparatus,

buffer solution and method employed are asfollows:

A column is charged wit'h a cation exchange resinas,

for example, with'a polystyrene sulfonic acid (H') cation exchange-resinof about. 200 to 400 mesh' such as BIORAD'A'G-50W-X8 400 mesh} l-l formresin. The column tubing, which contains a water jacket, consists ofpressure and stainless steel couplingsand connec-v tors. The top of thecolumn is provided with a threeway tap to allow for addition ofthebuffe'r following the. addition ,of the sample and thebottom ofthecolumn is provided with-a stream splitter so that the major portionof effluent is directed to an automatic fraction collector while aminute. portion is diverted to an automatic analyzing system. The columnis of suchsiz'e that an initial charge'of 5-l0 g. ofimixture of aminoacids can be separated. Aj s'mall portion of the effluent solutionis'continuou sl'y monitored viareaction with an ninhydrin reagent, i.e.,an'-- automatic analyzing system which 'is'equipped to maximizethesensitivityof the ninhydrin -response..The buffer solutions which areemployed in the-extraction of labelled'L arnino'acid are in general,volatilizable basic solutions having apH in the range of from about 3.0to '5 .0. Suitablebuffers include,

for example, pyridinium formate and. pyridinium '50 residue byhydrolysis with an acid as, for example, with e a mineral acid such ashydrochloric acid; humin and innon-.amino acid components and any:remaining acid are effectively eliminated. The adsorbed L-arnino acidsacetate which, underthe conditions oft-he reaction,,are

easily vaporized and serve to increase slightly the basicity. of thereaction mixture so as to make possible the separation of theindividuallylabelled products.v

Cuts of 25 .ml. each aretaken with a fraction collector and. appropriatecuts are pooled, concentrated in vacuo to dryness and excessaceticacidand pyridine is .removedby concentrating two additional times withwater. Partially deuterated L-aspartic-acid.(ll l), L-glutamic acid(Ill) and Latyrosine (ll) are collected and Y the recovered L-aminoacidsare identified by thinlayer silica gel-chromatography using'conventional systems and standards.

An alternate method for the separation of the par tially deuteratedL-tyrosine starting material (II) con.- sists intreating the deuteratedamino acid mixture obtained. according to the aforementioned processwith an aqueous solutionof an acid as,-forexample, with a mineral acidsuch as hydrochloric acid, maintained at'a Pl-l-of about 5.0 to 6.0followed by successive washings with suitableeluants such as water,ethanol, ethyl ether and the like. The partially deuteratedL-tyrosinea,,B,B

' ,2,6--d (11) thus obtained is in a purity suitable for use directly inthe process of this invention. The following'examples illustrate theprocess of this invention. Howeven-the 'examplesare illustrative onlyandany modification thereof which results in the formation of anidentical product should be construed as an analogous and functionallyequivalent method of preparation. 7

EXAMPLE 1 L-Tyrsoine-Carboxy,N,N,3 ,4,5- d

L-Tyrosine (1.0 g.) is heated under nitrogen in 6 N deuterium chloride(5ml.) at 1 10 C. for hours. The

deuterium chloride and deuterium-oxideare then removed by concentrationin vacuo and excess deuterium chloride is removed by repeating theconcentration in vacuo with additional water flushes. The resultingsolution of deuterated L-tyrosine is diluted to 50 m1., filtered and thefiltrate adjusted to pH 5.5 with the dropwise addition of 5 percentsodium hydroxide tional times in order to remove most of the freehydrochloric acid and the syrup is then dried in a vacuum ovencontaining a tray of sodium hydroxide pellets at a temperature of about50 C. for 16 hours in order to remove the last traces of freehydrochloric acid. It is critical to remove the last traces ofhydrochloric acid in order to obtain good adsorbtion on the resincolumn. The syrup is dissolved in 2.0 liters of water and filtreredthrough a sintered glass funnel to afford a filtrate comprising theamino acids and their hydrochloride salts and soluble impurities. Thevolume is adjusted to 3.0 liters. l

3. Resin Adsorbtion An 8.5 cm. diameter/80 cm. column (4.0 liters) isfilled with a s ulfonic acid ion exchange resin 8 percent cross linked(e.g., Dowex SO X8 resin) and converted to the hydrogen cycle. The resinis pretreated with 20 liters of 1.5 percent ammonium hydroxide solutionfollowed by the addition of 20 liters of water, whereafter 20 liters 'of10 percent hydrochloric acid solution is whereupon the partiallydeuterated L-tyrosine EXAMPLE 2 L-Tyrosine-d I Step A: Preparation ofDeuterated L-Amino Acid Mixture v g 1 l. Cell Rupture and ChlorophyllRemoval Lyophilized deuterated algae (250 g'.) is extracted with three3,500 m1. portions of 10' percent trichloroacetic. acidat 70 C. withagitation for 30 minutes. Upon cooling atroom temperature the algaeresidue is recovered by centrifugation at 5,000-'-10,000 g. for 10minutes at 5 l0 C. The crude protein residue is then agitated with 4.0liters of 22:1 ethanolethyl ether mixture and filtered on a largesintered glass funnel of medium porosity. The residue is washed againwith two 4-1iter' portions of solvent followed by washing with fivel-liter portions of ethyl ether. The residue is then dried in vacuo at30 C. to afford 130.0 g. of partially purified protein.

2. Hydrolysis The purified protein (1.26.5 g.) obtained according toStep A (1) is hydrolyzed in a sealed glass bomb by treatment with 1.800ml. of 6 N hydrochloric acid for 16 hours at 1 10 C. The hydrolysismixtureis saturated with a few drops of capryl alcohol to controlfoaming and is then cooled to room temperature and filtered through asintered glass funnel. The residue thus obtained is washed with three250 m1. portions of distilled water to afford amixture of amino acidscontaining an excess of hydrochloricacid. The residue is discardedandthe filtrate consisting of amino acids and excess hydrochloric acidis concentrated in vacuo at about 35 C. to afford a syrup and caprylalcohol is added to con-' I trolfoaming. Themixture is then redissolvedin 500 ml.

added to the mixture. The column is washed with distilled water at arate of 175 ml. per minute until free of chloride ion. The filteredhydrolysis solution obtained in Step A (2) containing the amino acid andtheir hydrochloride salts and soluble impurities is passed through thecolumn (60 ml. per minute) and washed with water until a volume of 12liters is collected. The column is then eluted with a 6 N ammoniumhydroxide solution. A void of 3,500 ml. is discarded and ten liters ofeluate are collected. In the mixture thus obtained'90 percent of theamino acids are present in the first two liters of eluate. The entirecollection of .eluates is concentrated in vacuo at about 35 C. to 500ml. and lyophilized to yield 81.3 gm. of'a mixture of deuterated L-aminoacids. 1

4. Deuterated L-Amino Acid Mixture .The mixture of deuterated L-aminoacids obtained according to Step A (3) is dissolved in 1.5 liters ofdistilled water and decolorized with an equal weight of decolorizingcarbon and stirred for one-half hour at of water and re'concentrated invacuo at about 35C. to

asyrup. The flushing with wa ter is repeated two addiroom temperature.The slurry is filtered over a thin layer of diatomaceous earth andwashed with two 500 ml. portions of water. The filtrate and washes arethen concentrated in vacuo to 500 ml. and lyophilized to afford 72 g. ofa white purified mixture of deuterated L- amino acids. The yield ofdeuterated L-amino acid mixture per g. of deuterated algae cells is 29.2percent; purity: 101.00 percent; moisture content: 7.1 1 percent.

Step B: L-Tyrosine-a,/3,B2,6-d and L-Tyrosine-Carboxy-N,N ,a,B,B,2,4,6-d

To a column charged with a sulfonic acid ion exchange resin [Biorad AG-50W-X8 (400'mesh) H form] is added the deuterated L-amino acid mixture(7.0 g.) obtained according to Step A in water (30 m1.) and adjusted toa pH of 2.2 with 6 N hydrochloric acid. The deuterated L-amino acidmixture obtained according toStep A is fed to the column at a rate of2.2 ml./minute and developed with a series of buffers beginning with a0.1 M pH 3.1 pyridinium formate buffer. Cuts of 25 ml. each are takenwith a fraction collector-and appropriate cuts are pooled, concentratedin vacuo to dryness and excess acetic acid an pyridine is removed byconcentrating two additional times with water.

tyrosinemolecule is deuterated.

. 7 I At the. en 'of cut 5.10 a 0.2 M pH 3.1 pyridinium formate' bufferi s fed to the column and 100 ml. cutsare' collected thereafter. Attheend ofcut 603 510.2 M pH Y 4.4 pyridiniurn acetate buffer is fed tothe column. Partially deuterated L-tyrosine (90mg), i.e., L-tyr osinewcarboxy-N,N,a,B,B,2,4,6 d in: which all hydrogen atoms exceptthose'bonded to the carbons'ortho to the hydroxy moiety are replaced bydeuterium, is collected overcuts 692-698. Thereadily exchangeable natureof the deuterium. atoms, bonded to the carbonyloxy,

hydroxy andramino moieties in aqueous solutionresluts in an isolationofjrthe said partially deuterated Lityrosine asL-tyrosine-afi,/3,2,6-d,,. Nuclear magnetic ,2,6 -d to be 198 percentdeutero pure.

resonance spectroscopy shows the. L tyrosinerqflfi urn. chloride (1ml;)'. There is thus obtained a substanfor the protonated L-glutamicacid reactant of Example and other wise followingthe procedurede'scribedtherein thereis thus obtained L-aspartic N,N,3,3-d

. EXA PLE? i v r DL-Aspartic-'N,N,3,3 d acid d I Protonated DL-asparti'cacid mg.) is heated .at

- 1-l5 C. for hours in 0.25 N potassium deuteroxide tially quantitativeamount of totally deuterated L- V tyrosine, namely,- L-tyrosine-dNuclear magnetic resonance spectroscopy indicates that the entire L-EXAMPLES Step'A': L'-Tyrosine-'a, [3,B,2,6-d The mixture of .partiallydeuterated L-amino acids (5.0 g.) obtainedaccording to Example 2, StepA, is

. dissolved in water ml.) and the pH adjusted to 5.5

with hydrochloric acid. toprecipitate partially deuterated LatyrosinewThe solution is stirred for 1 hour at 5C., filtered and-the precipitatewashed successively. withtwo 5 ml. portions of water, two 5m]. portionsof 'ethanolandtwo 5 ml. portions of ether. Upon drying the precipitatein vacuo at 60C. there is thus obtained 0. s.m1. under an atmosphere ofnitrogen. There is .thusobtaine'd DL aspartic-N,N,3,3,-d .acid-d tsubstituting protonated D L-tyr osine and protonated DL'glutamic acidfor DL-aspartic acid recited in theprecedingparagraph and following theprocedure described therein there is thusobtained DL- mg, of partiallydeuterated. L-tyrosine, i'.e.', L-

tyrosine-a,B,B,' 2,6 -d ,-in .which the carbon atomsortho 40'. v tothehydroxy radical are substituted by hydrogen. 7 Thinlayerchromatography on silica gel indicates an L- amino acid purityof morethan 95 percent Ltyrosinea, B, B, 2,6-d partially-deuterated L-tyrosine.

Step B: L-Tyros ine d i I The. L-tyrosine-a,fl,B,2,6-d mg.) obtainedaccording to Step'A 'isheated at 1 10 C. with N deuterium chloride (1ml.) There is thus obtained a substantially quantit ative amount oftotally deuterated. L-

magnetic tyrosine, i.e., L-tyrosine-d Nuclear resonance spectroscopyconfirms that the entire L- tyrosine molecule is deuterated.

EXAMPLE4 L-Tyrosine-Carboxy,N,N,3,4,5-d Protonated L-tyrosine 10 mg.) isheated at 110 C.

i for 20 hours in.,0.25 N sodium deuteroxide (0.5 ml.)

under a nitrogen. atmosphere. Nuclear ,magnetic resonance spectroscopyindicates that the product thus obtained isL-tyrosine-,carboxy,N,N,3,4,5-d

, EXAMPLE 5 L Glutamic-N'NAA-d; acid-d v I Y Protonated L-glutamic acid10 mg.) is heated in ,6 N

under, a. nitrogen. atmosphere. Nuclear magnetic tyro'sine-carboxy,N,N,3,4,5-d and DL-glutamic-v N;N,4,4-d acid,-d x 1 v v EXAMPLESL'-Aspartic Acid-d, f Step A':'L-Aspartic-2-d acid. To a column chargedwith a sulfonic acid ion exchange resin [Biorad"AG-50W-X8 (400,mesh),H.

form} is added the deuterated L-amino acid mixture (7.0 g.) obtainedaccording .to Example 1, Step ;.A, in

water (30 ml.) and adjustedto a pH of 2.2 with 6 N hydrochloric acid.The deuterated L-lamino acid mixture is fed to the column at a rate of2.2 ml./minute and .developed with a series e fbuffers beginning with a0.1 M pH 3.1 pyridinium format e buffer..;Cuts of 25 ml. each; are takenwith a fraction collector and ap-v propriat'e" cuts are pooled,concentrated in .vacuo to dryness andexcess acetic acid andpyridineisremoved by concentratingtwoadditional times with water.Partially'deuterated L-aspartic acid:(126' mg.), i.e., L? aspartic-Z-dacid," is collected over cuts"l56-'-183.

Nuclear magnetic resonance spectroscopy shows the L- deuterium chloride(0.5 m1.) at 115 C. for 20 hours aspartic-Z-d acid to be percent'deuteropure.

' Step B: L-Aspartic Acid-d By substituting the L-aspartic-2-d acidobtained accordingto Step .A for the L-tyrosineFa,B,B,2,6-'d reactantrecited in Example 2, Step'C, and following sub stantially the proceduredescribed therein there is thus obtained L-aspartic acid-d EXAMPLE 9L-Aspartic-N,-N,2-d 'acid-d L Aspartic acid-d 10 mg.) in 6 N'hydrochloric-acid (0.5 ml.) is heated at'll5 C. for'20 hours under'nitrogen. There is thus. obtained L-aspartic-Z-d acid, the structure ofwhich is'confirmed by nuclear:mag-, neticrcsonance'. spectroscopy.Treatment of the -L- aspartic-Zd acidwith deuterium oxide results in anexchange of the protons bonded to the amino and carboxy moieties bydeuteriumto afford L-aspartic-N,N ,2-

.d acid-d HYD' D II

wherein- R" is hydrogen or-deuterium.

2. A compoundaccording -to' claim 1 wherein the.

deuterated product is L-tyrosine having the structure:

wherein R is hydrogen or deuterium.

3. A compound according to claim 2 wherein the deuterated product isL-tyrosine-a,/3,B,2,6-d

4. A compound according to claim 1 wherein the deuterated product isL-tyrosine having the structure wherein R is hydrogen or deuterium.

5. A compound according to claim 4 wherein the deuterated product isL-tyrosine-carboxy,l l,N ,3,4,5-d 6. A method for effecting an exchangeof at least the X-hydrogen atoms'in the'L- or DL-products havingstructures 1 or II below I II by the reaction of ,said product I orproduct II with a deuterated inorganic acid or a deuterated metalhydroxide thus providing a product having structure Ill (from compoundI) orstructure W (from Compound ll) III IV wherein in each of theforegoing structures X represents hydrogen, R represents hydrogen ordeuterium and n represents 0 or the numeral 1.

7. The method according to claim 6 wherein the deuterated inorganic acidis a deuterated mineral acid.

8. The method of claim 6 wherein the deuterated metal hydroxide is analkali metal alkoxide deuteroxide or alkaline earth metal deuteroxide.

9. The method according to claim 7 wherein the deuterated mineral acidis employed in a concentration of from about 1 N to 7 N.

10The method according to claim 8 wherein the alkali metal deuteroxideor alkaline earth metal deuteroxide is employed in a concentration offrom about :11; The method according toclaim 6iwherein the process isconductedatthe reflux temperature of the reaction mixture. 1

12. A method according to claim 6 .for the preparation of selectivelydeuterated DL- or L-products having structure Ill-a or lV-a below III-apreparation of L-tyrosine-carboxy-N,N,3,4,5-d which comprisestreatingL-tyrosine with deuterium chloride at a temperature'in the range of fromabout -l 20 14. The method accordingto claim 12 for the preparation ofL-aspa'rtic-N,N,3,3-d acid-d which comprises treating. L-aspartic acidwith deuterium chloride at a'temperature in the range of from about 8012Q"*,,;

15. A method according to claim l2 for the preparation ofDL-aspartic-N,N,3,3-d acid-d which comprises treating DL-aspartic acidwith potassium deuteroxide at a temperature in the range of from about-1191C- v 16. The method according to claim 12 for the preparation ofL-glutaniic-N,N,4,4-d acid-d which comprises treating L-glutamic acidwith deuterium chloride at a temperature in the range of from aboutsflififlfllCw-w.

17. A method according to claim 6 for the preparation oftotallydeuteratedL-tyrosine, totally deuterated L-aspartic acid andtotally deuterated L-gl utarnic acid,

which comprises treating L-tyrosine-a,fl,,B,2,6-d L-L-tyrosinea,B,B,2,6-d with deuterium chloride at a,

temperature in the range of from about 80l 20 C.

- 11 I 7 19. The method 'according'to claim17 for the preparation ofL-aspartic acid-d which comprises treatingLeaS partic-Z-dacid-withdeuterium halide at a temperature in the range of from about 80 1 20:C.

20. A- method for the preparation of L-aspartic- -N,N,2-d acid-d 'andlJ-glutamic-N,N',2,3;3-d acid-d which-comprises treating .L-asparticacid-d r L-glutamic acid-d witha concentrated solution-of an acid atatemperature intherange of from about 90-l50 C.

I followed by thetreatment of the L-aspartic-Zd acid orL-gluta'mi'c-2,3,3-'d acid intermediate thus. obtainedwithvdeuteriumfloxidejto afford the desired product.

21." The method, according to claim 20 wherein the L=aspartic acidsdforL-glutamic acid-d is treated with mineral acidat a temperature in therange of from about l'l0--l20 C:

22; Theyme thod according to claim for the preparation ofL-aspartic-N,N,2d acid-d which comprises treating L-aspartic acid-d with 6 N-hydrochloric acid at a temperature of 1 15 C. to afford L-aspartie-2- dacid which, upon treatment with deuterium oxide, af-

which; comprisesthe reaction of compound I with a deuterated inorganicacid or a deuterated metalhydroxide thus providing a product having thestructure III whereinlin each of the foregoing: structures X representshydrogen and R represents hydrogen or deuterium. I

25. A methodfor, effecting exchange of atleast the X-hydrogen atoms inan L- or DL +compound having 7 the structure X NR2 I II whichcompris'esthe reaction of compound ll with a ,deuterated inorganic acid or adeu-terated metal hydroxide thus providing a product having thestructure wherein in each of the foregoingvstructures X irepresentshydrog'en, R represents hydrogen or deuterium, andn-represents 0 or the numeral 1.

2. A compound according to Claim 1 wherein the deuterated product isL-tyrosine having the structure:
 3. A compound according to Claim 2wherein the deuterated product is L-tyrosine- Alpha , Beta , Beta,2,6-d5.
 4. A compound according to claim 1 wherein the deuteratedproduct is L-tyrosine having the structure
 5. A compound according toClaim 4 wherein the deuterated product isL-tyrosine-carboxy,N,N,3,4,5-d6.
 6. A method for effecting an exchangeof at least the X-hydrogen atoms in the L- or DL-products havingstructures I or II below
 7. The method according to claim 6 wherein thedeuterated inorganic acid is a deuterated mineral acid.
 8. The method ofclaim 6 wherein the deuterated metal hydroxide is an alkali metalalkoxide deuteroxide or alkaline earth metal deuteroxide.
 9. THe methodaccording to claim 7 wherein the deuterated mineral acid is employed ina concentration of from about 1 N to 7 N.
 10. The method according toclaim 8 wherein the alkali metal deuteroxide or alkaline earth metaldeuteroxide is employed in a concentration of from about 0.1 N to about1.0 N.
 11. The method according to claim 6 wherein the process isconducted at the reflux temperature of the reaction mixture.
 12. Amethod according to Claim 6 for the preparation of selectivelydeuterated DL- or L-products having structure III-a or IV-a below 13.The method according to claim 12 for the preparation ofL-tyrosine-carboxy-N,N,3,4,5-d6, which comprises treating L-tyrosinewith deuterium chloride at a temperature in the range of from about80*-120* C.
 14. The method according to claim 12 for the preparation ofL-aspartic-N,N,3,3-d4 acid-d2, which comprises treating L-aspartic acidwith deuterium chloride at a temperature in the range of from about80*-120* C.
 15. A method according to claim 12 for the preparation ofDL-aspartic-N,N,3,3-d4 acid-d2, which comprises treating DL-asparticacid with potassium deuteroxide at a temperature in the range of fromabout 80*-110* C.
 16. The method according to claim 12 for thepreparation of L-glutamic-N,N,4,4-d4 acid-d2, which comprises treatingL-glutamic acid with deuterium chloride at a temperature in the range offrom about 80*-120* C.
 17. A method according to claim 6 for thepreparation of totally deuterated L-tyrosine, totally deuteratedL-aspartic acid and totally deuterated L-glutamic acid, which comprisestreating L-tyrosine- Alpha , Beta , Beta ,2,6-d5, L-aspartic-2-d acid orL-glutamic-2,3,3-d3 acid with a deuterated mineral acid or an alkalimetal deuteroxide or alkaline earth metal deuteroxide.
 18. The methodaccording to claim 17 for the preparation of L-tyrosine-d11, whichcomprises treating L-tyrosine- Alpha , Beta , Beta ,2,6-d5 withdeuterium chloride at a temperature in the range of from about 80*-120*C.
 19. The method according to claim 17 for the preparation ofL-aspartic acid-d7, which comprises treating L-aspartic-2-d acid withdeuterium halide at a temperature in the range of from about 80*-120* C.20. A method for the preparation of L-aspartic-N,N,2-d3 acid-d2 andL-glutamic-N,N,2,3,3-d5 acid-d2, which comprises treating L-asparticacid-d7 or L-glutamic acid-d9 with a concentrated solution of an acid ata temperature in the range of from about 90*-150* C. followed by thetreatment of the L-aspartic-2d acid or L-glutamic-2,3,3-d3 acidintermediate thus obtained with deuterium oxide to afford the desiredproduct.
 21. The method according to claim 20 wherein the L-asparticacid-d7 or L-glutamic acid-d9 is treated with mineral acid at atemperature in the range of from about 110*-120* C.
 22. The methodaccording to claim 20 for the preparation of L-aspartic-N,N,2-d3acid-d2, which comprises treating L-aspartic acid-d7 with 6 Nhydrochloric acid at a temperature of 115* C. to afford L-aspartic-2-dacid which, upon treatment with deuterium oxide, affords the desiredproduct.
 23. The method according to claim 20 for the preparation ofL-Glutamic-N,N,2,3,3-d5 acid-d2, which comprises treating L-glutamicacid-d9 with 6 N hydrochloric acid at 115* C. to affordL-glutamic-2,3,3-d3 acid which, upon treatment with deuterium oxide,affords the desired product.
 24. A method for effecting exchange of atleast the X-hydrogens or L- or DL-tyrosine having the structure
 25. Amethod for effecting exchange of at least the X-hydrogen atoms in an L-or DL-compound having the structure